Present embodiments of the invention relate to a Type II restriction endonuclease, Acinetobacter calcoaceticus 65 (Acc65I), obtainable from the recombinant strain carrying the genes for the Acc65I restriction-modification system from Acc65I in E. coli, and to a process for producing the same.
Restriction endonucleases are enzymes that occur naturally in certain unicellular microbes—mainly bacteria and archaea—and that function to protect these organisms from infections by viruses and other detrimental DNA elements. Restriction endonucleases bind to specific nucleotide (nt) sequences in double-stranded DNA molecules (dsDNA) and cleave the DNA molecules, often within or close to these sequences, fragmenting the molecules and triggering their ultimate destruction. Restriction endonucleases commonly occur with one or more companion enzymes termed modification methyltransferases. The methyltransferases bind to the same nt sequence in dsDNA as the restriction endonuclease, but instead of cleaving the DNA, they modify it by the addition of a methyl group to one of the bases in each strand of the sequence. This modification prevents the restriction endonuclease from binding to that site thereafter, effectively rendering the site resistant to cleavage. Methyltransferases act as cellular antidotes to the restriction endonucleases they accompany, protecting the DNA of the cell from destruction by its own restriction endonucleases. Together, a restriction endonuclease and its companion modification methyltransferase(s) form a restriction-modification (R-M) system, an enzymatic partnership that accomplishes for microbes, to some extent, what the immune system accomplishes for multicellular organisms.
A large and varied group of restriction endonucleases termed ‘type II’ cleave DNA at defined positions, and can be used in the laboratory to cut DNA molecules into precise fragments for gene cloning and analysis. The biochemical precision of type II restriction endonucleases far exceeds anything achievable by chemical methods, and so these enzymes have become the reagents sine qua non of modern molecular biology. They are the ‘scissors’ by means of which genetic engineering and analysis is performed, and their adoption has profoundly impacted the biomedical sciences over the past 25 years, transforming the academic and commercial arenas, alike. Their utility has spurred a continuous search for new restriction endonucleases, and a large number have been found. Today more than 200 Type II endonucleases are known, each possessing different DNA cleavage characteristics (Roberts and Macelis, Nucl. Acids Res. 29:268-269 (2001)); (REBASE®, http://rebase.neb.com/rebase). Concomitantly, the production and purification of these enzymes have been improved by the cloning and over-expression of the genes that encode them in non-natural production strain host cells such as E. coli. 
Since the various restriction enzymes perform similar roles in nature, and do so in much the same ways, it might be thought that they would resemble one another closely in amino acid sequence, organization, and behavior. Experience shows this not to be true, however. Surprisingly, far from resembling one another, most Type II restriction enzymes appear unique, resembling neither other restriction enzymes nor any other known kind of protein. Type II restriction endonucleases seem to have arisen independently of one another for the most part during evolution, and to have done so hundreds of times, so that today's enzymes represent a motley collection rather than a discrete family. Some restriction endonucleases act as homodimers, some as monomers, others heterodimers. Some bind symmetric sequences, others asymmetric sequences; some bind continuous sequences, others discontinuous sequences; some bind unique sequences, others multiple sequences. Some are accompanied by a single methyltransferase, others by two, and yet others by none at all. Often the methyltransferase is separate from, and functions independently of, the restriction endonuclease, but sometimes the two are fused and interdependent. When two methyltransferases are present, on some occasions they are separate proteins, on others they are fused. The orders and orientations of restriction and modification genes vary, with all possible organizations occurring. Several kinds of methyltransferases exist, some methylating adenines (m6A-MMases), others methylating cytosines at the N-4 position (m4C-MMases), or at the 5 position (m5C-MMases). Usually there is no way of predicting, a priori, which modifications will block a particular restriction endonuclease, which kind(s) of methyltransferases(s) will accompany that restriction endonuclease in any specific instance, nor what their gene orders or orientations will be.
From the point of view of cloning a Type II restriction endonuclease, the great variability that exists among restriction-modification systems means that, for experimental purposes, each is unique. Each enzyme is unique in amino acid sequence and catalytic behavior; each occurs in unique enzymatic association, adapted to unique microbial circumstances; and each presents the experimenter with a unique challenge. Sometimes a restriction endonuclease can be cloned and over-expressed in a straightforward manner but more often than not it cannot, and what works well for one enzyme can work not at all for the next. Success with one is no guarantee of success with another.
There is a continuing need for novel type II restriction endonucleases. Although type II restriction endonucleases that recognize a number of specific nucleotide sequences are currently available, new restriction endonucleases that recognize novel sequences provide greater opportunities and ability for genetic manipulation. Each new unique endonuclease enables scientists to precisely cleave DNA at new positions within the DNA molecule, with all the opportunities this offers.